This project is concerned with the elucidation of the mechanism of the mammalian heart mitochondrial and Rhodospirillum rubrum energy-linked pyridine dinucleotide transhydrogenase reaction: NADH plus NADP ion plus x-y yields (reversibly) NAD ion plus NADPH plus x plus y where x-y is a high-energy intermediate or state of the oxidative phosphorylation system. Problems to be investigated with the bovine heart enzymes are: (a) localization of the transhydrogenase in the inner mitochondrial membrane; (b) stoichiometry of proton translocation coupled to transhydrogenation in submitochondrial particles; (c) pyridine dinucleotide and energy dependent conformational alterations of the enzyme; (d) demonstration of a possible reduced enzyme intermediate; (e) elucidation of amino acid residues of the enzyme involved in proton translocation; (f) purification of bovine heart transhydrogenase and reconstitution of energy-linked transhydrogenation by incorporation of the purified enzyme into proteoliposomes. Further studies on the R. rubrum transhydrogenase include: (a) purification of both the soluble component and the membrane component; (b) determination of the specificity and locale of the substrate binding sites on the two enzyme components; (c) determination of the component containing a reduced enzyme intermediate; and (d) further elucidation of NADP(H) induced conformational changes of the membrane bound component.